Abstract Body: Introduction: Atherosclerosis is characterized by the accumulation of lipoproteins and immune cells, leading to unresolved inflammation in the arterial wall. Endothelial cells (EC) form a barrier, and modulate communication, between circulating and resident vascular cells. EC dysfunction, a hallmark of the disease, disrupts healthy communication. Extracellular vesicles (EVs) are key mediators of intercellular communication, carrying bioactive molecules that influence cell function and signal disease states. While our group previously demonstrated crosstalk between endothelial-derived EVs (EC-EVs) with monocytes and smooth muscle cells in vitro, their role in vivo remains unclear. To address this, we developed a murine model to detect and visualize EC-EVs in atherosclerosis.

Methods: We generated an EC-EV tracking model on an Ldlr-/- background using an inducible endothelial-specific Cre to activate the expression of an EV-enriched CD63 protein fused with emerald GFP. Mice (Ldlr^-/- Cdh5Cre^ERT2+/- CD63-emGFP⁺) aged 8-10 weeks, and Cre^- control, received tamoxifen to induce GFP⁺EC-EVs. Mice were fed a standard chow or high-cholesterol diet for 12 weeks to induce atherosclerosis. EVs were isolated from the aorta and heart, quantified, and characterized by nanoparticle tracking analysis (NTA) and western blot. EC-EVs were visualized in aortic sections via confocal microscopy and immunogold-labelled transmission electron microscopy (TEM).

Results: EVs, characterized by EV markers (CD63, Alix and CD9) and the absence of Calnexin, were isolated from the aorta and heart of chow-fed mice and quantified by NTA. CD63-emGFP was detected exclusively in Cre⁺ mice. Confocal imaging of aortic root sections showed a GFP⁺ signal in luminal and adventitial ECs of Cre⁺ mice, with no GFP in Cre^- control. Immunogold TEM confirmed GFP⁺ EVs in ECs of Cre⁺ mice. Confocal and TEM imaging revealed EC-EV trafficking in atherosclerotic plaque, with EC-EVs (anti-GFP) localizing with ECs (anti-CD31) and within plaques in CD31 negative cells. Co-staining for GFP and CD68 indicated EC-EVs in macrophages, suggesting communication with myeloid cells. TEM confirmed EC-EVs (GFP gold particles) inside plaque macrophages.

Conclusion: This is the first in vivo approach to detect EC-EV trafficking within the atherosclerotic vessel wall. Understanding EC communication in atherosclerosis will aid in the development of new clinical biomarkers and novel therapeutic strategies for vascular diseases.